FUNCTIONAL ANALYSIS OF DENGUE VIRUS ANTIGENS NS3 AND NS5

描述(改编自申请人的摘要)：本报告的总体目标 建议阐明登革病毒抗原NS3和NS3的作用 NS5参与蛋白质加工和基因组复制。登革热是人类 由蚊媒登革热病毒引起的疾病。与登革热分离 发烧是一种更严重和致命的疾病，发生在儿童中。 被称为登革出血热，或登革热休克综合征。一个 与发展战略有关的潜在问题 单价登革热疫苗是指感染一种血清型的人 完全容易被其他人感染。这种继发性感染 可能导致严重和致命的疾病形式。一种方法是 控制病毒感染是为了了解病毒的复制策略 这样就可以开发出一种抗病毒药物。病毒式传播 抗原NS3和NS5是此类研究的有用靶标，因为它们 是高度保守的黄病毒非结构蛋白，它们已经被 与蛋白质加工和病毒复制有关。展示了NS3 在前180个氨基酸中包含一个丝氨酸蛋白酶结构域。 与该结构域相邻的区域与几个RNA相似 解旋酶和复制蛋白。NS5含有Gly-Asp基序- 天冬氨酸，常见于几种依赖RNA的RNA聚合酶中。 NS3RNA解旋酶是否在启动中起作用的问题 通过融化高度保守和稳定的次级病毒 将解决在DEN-2 RNA的3‘端发现的结构。离体 含有相同序列和极性的DEN-2 RNA的转录本，如 作为模型，双链RNA底物将被用于 确定NS3解旋酶的特性，然后进行定点突变 推测的解旋酶结构域。NS5 RNA聚合酶可能有可能 在病毒负链合成中发挥作用。模型RNA 将使用底物作为模板/引物来表征 NS5的合成活性。Gly-Asp-Asp的定点突变 将进行NS5结构域的研究，以检测其在RNA中的功能作用 综合。登革热病毒的所有非结构蛋白都已经 利用重组痘苗病毒系统进行表达。同样， 所有的结构蛋白都将得到表达。对蛋白质进行解剖 以及起始和延伸的核苷酸序列要求 复制、缺陷干扰(DI)构建中的步骤，包括 5‘-和3’-非编码的RNA基因组将在 结构蛋白和非结构蛋白。 了解多蛋白前体的加工机制， 它能产生NS3和NS5等成熟蛋白质，是脱落的关键 基因组复制所需的蛋白质。证据来自 我们的实验室认为，NS2B是NS3发挥作用所必需的 NS3-&NS5前驱体加工中的蛋白酶 反式乳沟。NS2B和NS3将被提纯为同质 常规和亲和柱层析方法。几个 含有二碱基残基和蛋白酶的底物类似物 将使用抑制剂对纯化的NS2B/NS3酶进行表征。

DESCRIPTION (Adapted from applicant's abstract): The overall aim of this proposal is to elucidate the role of the dengue viral antigens NS3 and NS5 in protein processing and genome replication. Dengue is a human disease caused by the mosquito-borne dengue virus.Apart from dengue fever, a more severe and fatal form of the disease occurs in children called dengue hemorrhagic fever, or the dengue shock syndrome. A potential problem associated with the strategy for the development of monovalent dengue vaccines is that individuals infected with one serotype are fully susceptible to infection by others. This secondary infection may result in the severe and fatal form of the disease. One approach to control the virus infection is to understand the replication strategy of the virus so that an antiviral agent can be developed. The viral antigens, NS3 and NS5, are useful targets for such studies, because they are highly conserved flavivirus nonstructural proteins, which have been implicated in protein processing and viral replication. NS3 was shown to contain a serine protease domain within the first 180 amino acids. Adjacent to this domain, there are regions of similarity to several RNA helicases, and to replication proteins. NS5 contains the motif Gly-Asp- Asp, which is often found in several RNA-dependent RNA polymerases. The question of whether the NS3 RNA helicase plays a role in initiation of viral replication by melting the highly conserved and stable secondary structure found at the 3'-end of DEN-2 RNA will be addressed. In vitro transcripts containing the same sequence and polarity of DEN-2 RNA, as well as model double-stranded RNA substrates will be used to characterize the NS3 helicase, followed by site-directed mutagenesis of the putative helicase domains. The NS5 RNA polymerase could potentially play a role in the viral negative strand synthesis. Model RNA substrates as template/ primers will be used to characterize the synthetic activity of NS5. Site-directed mutagenesis of the Gly-Asp-Asp domain of NS5 will be carried out to examine its functional role in RNA synthesis. All the nonstructural proteins of dengue virus have already been expressed using the recombinant vaccinia virus system. Similarly, all the structural proteins will be expressed. To dissect the protein and the nucleotide sequence requirements for initiation and elongation steps in replication, defective-interfering (DI) constructs containing the 5'-and 3'-noncoding RNA genome will be used in the presence of structural and nonstructural proteins. Understanding the mechanism of processing of the polyprotein precursors, which generates mature proteins such as NS3 and NS5, is critical to shed light on the protein requirements for genome replication. Evidence from our laboratory suggests that NS2B is required for NS3 to function as a protease in the processing of NS3->NS5 precursor involving both cis and trans cleavages. NS2B and NS3 will be purified to homogeneity using conventional as well as affinity column chromatographic methods. Several substrate analogues containing dibasic residues, as well as protease inhibitors will be used to characterize the purified NS2B/NS3 protease.